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  1. Abstract

    Cryopreservation by vitrification has far-reaching implications. However, rewarming techniques that are rapid and scalable (both in throughput and biosystem size) for low concentrations of cryoprotective agent (CPA) for reduced toxicity are lacking, limiting the potential for translation. Here, we introduce a joule heating–based platform technology, whereby biosystems are rapidly rewarmed by contact with an electrical conductor that is fed a voltage pulse. We demonstrate successful cryopreservation of three model biosystems with thicknesses across three orders of magnitude, including adherent cells (~4 µm),Drosophila melanogasterembryos (~50 µm) and rat kidney slices (~1.2 mm) using low CPA concentrations (2–4 M). Using tunable voltage pulse widths from 10 µs to 100 ms, numerical simulation predicts that warming rates from 5 × 104to 6 × 108 °C/min can be achieved. Altogether, our results present a general solution to the cryopreservation of a broad spectrum of cellular, organismal and tissue-based biosystems.

     
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  2. Abstract

    The development of a widely adopted cryopreservation method remains a major challenge inDrosophilaresearch. Here we report a robust and easily implemented cryopreservation protocol ofDrosophila melanogasterembryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.

     
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